Compositions and methods for reproducing and modulating mammalian messenger RNA decapping

ABSTRACT

An in-vitro system in which mammalian messenger RNA decapping occurs is provided, for use in identifying modulators, deficiencies, and other aspects of the regulation of RNA turnover.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. 119(e) from U.S. Ser.No. 60/233,682, filed Sep. 19, 2000, which is incorporated herein byreference in its entirety.

GOVERNMENTAL SUPPORT

The research leading to the present invention was supported, at least inpart, by Grant Nos. GM58276, GM6382-01, and CA80062 from the NationalInstitutes for Health. Accordingly, the Government may have certainrights in the invention.

BACKGROUND OF THE INVENTION

The steady state level of a messenger RNA (mRNA) is a function of itsrate of synthesis and degradation. Just as numerous differences in therate of synthesis (i.e., transcription) exist, so to do differences inrelative mRNA stability. Messenger RNAs with very short half-livesinclude many clinically important transcripts, namely transcriptionfactors, growth factors and cytokines. Messenger RNAs with extremelylong half-lives also have been described, including globin transcripts.In addition, messenger RNAs from some disease alleles contain aninappropriately placed nonsense codon resulting in premature terminationof the open frame encoding the protein product. These “nonsensecodon-containing” mRNAs are degraded very rapidly in cells. Clearly,MRNA stability plays an important regulatory role in the molecularbiology of the cell.

Since mRNA stability plays a key role in regulated gene expression, itis a natural target for drug development. This potential for drugdevelopment is further enhanced when one surveys some of the bestdescribed cases of regulated MRNA stability. First, the importance ofmodulation of the immune system is central to many aspects of clinicalmedicine. Several genes that play a key role in immunologic responsesand development (e.g. cytokines) are clearly regulated at the level ofmRNA stability. Second, the expression of several proteins that playregulatory roles in the cell cycle are also regulated at the level ofmRNA stability. This observation implies that MRNA stability mayinfluence cancer treatments, for example regulation of MRNA turnover cancontrol the expression of a variety of growth factors andproto-oncogenes. Finally, all aspects of gene therapy would benefit froma better understanding of modulating influences of mRNA stability.Messenger RNA stability, therefore, remains an unexploited target fordrug development with a great deal of potential.

Messenger RNAs contain a poly(A) tail of approximately 200 bases attheir 3′ end when they are synthesized. The first step in the pathway ofturnover of mRNAs in mammalian cells appears to be the stepwise removalof the tail by a process called deadenylation. The rate ofdeadenylation, as well as the overall rate of turnover of the MRNA, isinfluenced by sequence elements present in the MRNA. The bestcharacterized of these elements to date is an “AU rich” element locatedin the 3′ untranslated region of the mRNA body. The presence of an AUrich element in a mRNA causes a dramatic increase in the rate ofdeadenylation/degradation, effectively shortening the half-life of amRNA. After the poly(A) tail is shortened to a minimal length, the bodyof the transcript is rapidly degraded with no apparent intermediates.Certain aspects of the foregoing are described in co-pending applicationSer. No. 09/320,609, filed May 26, 1999, incorporated herein byreference in its entirety.

In the yeast Saccharomyces cerevisiae, deadenylated mRNAs are usuallydegraded by two major pathways. In the predominant pathway, deadenylatedmRNAs are decapped and degraded by a 5′-to-3′ exonuclease. If thispathway is blocked by genetic mutations, an alternative pathway can beobserved in which deadenylated mRNAs are degraded by a 3′-to-5′exonuclease.

It is not known how mammalian mRNAs are degraded in cells followingtheir deadenylation nor how the process is regulated. Only one study hasbeen performed to date that suggests the possibility that mammalianmRNAs can be decapped in vivo. This study, however, used an indirecthighly-sensitive PCR assay that may not have detected true intermediatesin the mRNA degradation pathway. There is currently no directbiochemical evidence for decapping in mammalian cells. The putativehuman homologue of the yeast decapping enzyme Dcp1p surprisingly doesnot possess detectable decapping activity and has recently beenidentified as a transcription factor.

The lack of mechanistic detail in the model for MRNA turnover presentedabove reflects the lack of a good experimental approach to study theprocess. Human cells and mammals in general do not represent a geneticsystem that is easily exploited using current technology. The key tounderstanding mechanisms of gene expression in cells from higherorganisms, therefore, lies in a biochemical approach. When used inconjunction with reagents developed from chemical or molecularapproaches, such systems can provide the backbone of assays tounderstand and exploit aspects of cellular biology for therapeuticadvantages.

Decapping is a major regulated step in the turnover of yeast mRNAs. Itis a key regulatory element in mammalian cells as well. As shown herein,AU-rich instability elements stimulate decapping efficiency. Severalother elements have been identified in mammalian mRNAs that stabilizetranscripts. A 51 base pyrimidine-rich element has been identified inthe relatively stable alpha-globin MRNA that is responsible for itsextremely long half-life in vivo. As shown herein, such stabilityelements also regulate decapping efficiency. Regulation of mRNAdecapping, therefore, plays an important role in the posttranscriptional regulation of gene expression.

It is toward the development of an in vitro system in which mammalianmessenger RNA decapping occurs and can be exploited for the screening ofdecapping modulators, for identifying species-specific decapping enzymesand associated factors, among other uses, that the present invention isdirected.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication.

SUMMARY OF THE INVENTION

In one broad aspect, the invention is directed to a compositioncomprising:

-   -   a) a mammalian cell cytoplasmic extract;    -   b) a methylated cap analog; and    -   c) a cap-labeled mRNA substrate.

The composition provides the necessary components for observingdecapping of mammalian mRNA in vitro. The mammalian cell cytoplasmicextract may be, for example, an S100 extract comprising 100,000×g, 1hour supernatant from a mammalian cell lysate, the cells obtained frommammalian cells or tissue, including cell culture. In one embodiment,HeLa cells (human cervical cancer cell line) are used. The extract maybe prepared by dialysis of a said extract containing 10% glycerol. AnS100 extract is one example of an extract, and is useful in eachembodiment of this invention, although other mammalian cell cytoplasmicextracts are also contemplated.

The methylated cap analog may be, for example, ^(7me)GpppG or ^(7me)GTP.

The cap-labeled MRNA substrate may be detectably labeled at the alphaphosphate of the cap, and may be a label such as a radioactive label, anon-radioactive isotopic label, a fluorescent moiety, avisibly-detectable moiety, a releasable substrate or a co-factor for achemical or enzymatic reaction. The cap-labeled mRNA substrate mayinclude poly(A) or at least one RNA element which can be an instabilityelement, such as an AU-rich element, or a stability element, such as apyrimidine-rich element.

In another embodiment, the aforementioned composition may be used toadditionally detect MRNA deadenylation and degradation. The mammaliancell cytoplasmic extract may be depleted of activity of proteins thatbind polyadenylate.

In a further embodiment, the invention is directed to a kit for in-vitromammalian mRNA decapping containing at least:

-   -   a) a mammalian cell cytoplasmic extract; and    -   b) a methylated cap analog.

The kit may further include a cap-labeled MRNA substrate, for example,one labeled at the alpha phosphate of the cap. The label may be aradioactive label, a non-radioactive isotopic label, a fluorescentmoiety, a visibly-detectable moiety, a releasable substrate or aco-factor for a chemical or enzymatic reaction.

In another embodiment, the mammalian cell cytoplasmic extract may bedepleted of activity of proteins that bind polyadenylate.

The invention is also directed to a method for carrying out in vitromammalian mRNA decapping including at least the steps of

-   -   a) providing any of the compositions as described above        containing at least a mammalian cell cytoplasmic extract, a cap        analog and a cap-labeled mRNA substrate;    -   b) incubating the composition at about 30° C. for about 30 min        and monitoring decapping by detection of release of label from        said cap-labeled RNA.

In another embodiment, a method is provided for identifying a compoundas a modulator of mammalian mRNA decapping comprising carrying out theabove method in the presence and absence of said compound, andcorrelating any change in decapping by the presence of said compoundwith modulator activity of said compound.

In either of the foregoing methods, the cap-labeled mRNA substrate mayinclude poly(A) or at least one RNA element which can be an instabilityelement, such as an AU-rich element, or a stability element, such as apyrimidine-rich element.

Also part of this invention is a polypeptide with the following uniquecombination of properties: a molecular weight of about 50 to about 100kilodaltons (kD) in molecular exclusion chromatography; precipitation by20% ammonium sulfate, elution at between about 440 to 500 mM NaCl from aheparin-Sepharose column, and the ability to decap mammalian RNA. Apolynucleotide encoding the polypeptide and antibody which binds to thepolypeptide are also contemplated.

These and other aspects of the present invention will be betterappreciated by reference to the following drawings and DetailedDescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Identification of a decapping activity in HeLa cytoplasmicextracts. Panel A. SVARE-A0 RNA, radiolabeled exclusively at thealpha-phosphate of the 5′ cap structure, was incubated in standarddecapping conditions using no extract, S. cerevisiae whole cell extract(yeast lane), or HeLa S100 cytoplasmic extract. The ^(7me)GDP product ofthe decapping reaction was resolved by thin layer chromatography on PEIcellulose sheets developed using 0.45 M ammonium sulfate. Theidentification of radioactive spots was determined using markers thatwere visualized by UV shadowing. Panel B. GemARE-A60 RNA, radiolabeledexclusively at the alpha-phosphate of the 5′ cap structure, wasincubated in the absence of extract (no extract lane) or in the presenceof three independently prepared S100 cytoplasmic extracts from HeLacells (A, B, or C) either in the absence (− lanes) or presence of 20micromolar cap analog (+ lanes). The ^(7me)GDP product of the decappingreaction was resolved by chromatography on PEI cellulose sheetsdeveloped using 0.45 M ammonium sulfate.

FIG. 2. Methylated cap analog specifically activates decapping in HeLacytoplasmic extracts by sequestering cap-binding proteins. Panel A. Theindicated amounts of ^(7me)GpppG or GpppG were incubated with HeLa S 100extracts and decapping assays were performed using cap-labeledGemARE-A60 RNA. The products of decapping were analyzed on PEI-cellulosesheets. The arrowhead indicates ^(7me)GDP. Panel B. The indicatedamounts of ^(7me)GpppG or GpppG were incubated in HeLa S 100 extractsusing cap-labeled GemARE-A60 RNA under decapping conditions. After 5min., UV cross-linking was performed, mixtures were treated withribonuclease, and proteins radiolabeled through cross-linking tocap-labeled RNA oligomers were analyzed by electrophoresis on a 15%acrylamide gel containing SDS. The position of eIF4E is indicated by thearrowhead. Panel C. The indicated amounts of ^(7me)GpppG or GpppG wereincubated in HeLa S 100 extracts using cap-labeled GemARE-A60 RNA underdecapping conditions. After 5 min., UV cross-linking was performed,mixtures were treated with ribonuclease, DAN/PARN proteins radiolabeledthrough cross-linking to cap-labeled RNA oligomers wereimmunoprecipitated and analyzed by electrophoresis on a 10% acrylamidegel containing SDS. The position of DAN/PARN is indicated by thearrowhead. Panel D. The indicated amounts of ^(7e)GpppG or GpppG wereincubated in HeLa S100 extracts using cap-labeled GemARE-A0 RNA underdecapping conditions. The top panel shows UV cross-linking analysis oftotal protein as described in B to identify eIF4E. The middle panelshows UV cross-linking/immunoprecipitation analysis as described in C toidentify DAN/PARN. The bottom panel shows the products of a decappingassay that were analyzed by thin layer chromatography as described in A.

FIG. 3. The presence of a poly(A) tail represses decapping of threeindependent RNA substrates. Equimolar amounts of three independent,cap-labeled RNA substrates that either lacked a poly(A) tail (GemARE-A0,SVARE-A0 and GM-CSFT-A0) or contained 60 adenylate residues at their 3′end (GemARE-A60, SVARE-A60 and GM-CSFT-A60) were incubated in the invitro decapping assay in the presence of cap analog. Aliquots wereremoved at the indicated time points and reaction products were analyzedby thin layer chromatography on PEI cellulose sheets.

FIG. 4. The addition of poly(A) competitor RNA specifically activatesdecapping of polyadenylated RNA substrates. Panel A. Cap-labeledGemARE-A60 RNA, which contained 60 adenylate residues at its 3′ end, wasincubated in the in vitro decapping system for 30 minutes in thepresence of cap analog and the indicated amount of cold poly(A)competitor RNA. Reaction products were analyzed by thin layerchromatography on PEI cellulose sheets. In the lane marked GemARE-A0,GemARE-A0 RNA (that lacks a poly(A) tail) was incubated in the in vitrodecapping assay in the absence of poly(A) RNA competitor. Panel B.Cap-labeled GemARE-A60 RNA, was incubated in the in vitro decappingsystem for 30 minutes in the presence of cap analog and the indicatedamount of cold poly(A) or poly (C) competitor RNAs. Reaction productswere analyzed by thin layer chromatography on PEI cellulose sheets.Input RNA was run in the lane designated input. Panel C. Cap-labeledGemARE-A0 RNA, which lacked a poly(A) tail, was incubated in the invitro decapping system for 30 minutes in the presence of cap analog andthe indicated amount of cold poly(A) competitor RNA. Reaction productswere analyzed by thin layer chromatography on PEI cellulose sheets. Forall three panels, the position of ^(7me)GDP is indicated by anarrowhead.

FIG. 5. The presence of an AU-rich element significantly stimulates theefficiency of decapping. Panels A and B. A matched pair of cap-labeledRNA substrates that either lacked (Gem-A0 or SV-A0) or contained theTNF-alpha AU-rich element (GemARE-A0 or SVARE-A0) were incubated in thein vitro decapping system in the presence of cap analog for theindicated amount of time. Reaction products were analyzed by thin layerchromatography on PEI cellulose sheets. Panel C. A matched pair ofcap-labeled RNA substrates that either lacked (GM-CSF(-ARE)) orcontained the GM-CSF AU-rich element (GM-CSF (+ARE)) were incubated inthe in vitro decapping system in the presence of cap analog for theindicated amount of time. Reaction products were analyzed by thin layerchromatography on PEI cellulose sheets. For all three panels, theposition of ^(7me)GDP is indicated by an arrowhead.

FIG. 6. The stimulation of decapping by AU-rich elements requiressequence-specific AU-rich element binding factors. Cap-labeled GemARE-A0RNA (Panel A) or Gem-A0 RNA (Panel B) was incubated in the in vitrodecapping system in the presence of cap analog and the indicated amountof a 34 base synthetic RNA competitor derived from the TNF-alpha AU-richelement (ARE oligo lanes) or a 33 mer derived from randomly selectedsequences (non-specific oligo lanes). Reaction products were analyzed bythin layer chromatography on PEI cellulose sheets. The position of^(7me)GDP is indicated by an arrowhead. The numbers at the bottom referto decapping efficiency relative to lane 0.

FIG. 7: A model for the regulated mRNA decapping. The mRNA cap structureis normally stabilized by interactions with eIF4E and/or a novel PABPcomplex. The process of translation or the action of AU-rich elementbinding proteins can disrupt these complexes involving the 5′ cap,exposing the ends of the transcript to the deadenylation machinery.Following poly(A) tail shortening, the affinity of DAN/PARN for the mRNAis dramatically reduced, allowing the decapping enzyme (as well as otherdegradative enzymes such as 3′-to-5′ exonucleases) access to the ends ofthe mRNA.

FIG. 8: Partial purification of decapping activity from HeLa cytoplasmicextract. Sheet 1: Panel A. Ammonium sulfate fractionation of decappingactivity in Hela S 100. Sheet 2: Panel B. Chromatographic profile ofdecapping activity on a Superose-6 column. Panel C. Chromatographicprofile of decapping activity on a Heparin-Sepharose column.

FIG. 9: The mRNA stabilizing element from the alpha-globin generepresses decapping in vitro. Cap labeled GemA0 RNA or a variantcontaining the 51 base alpha-globin stability element (Gem-aGlob-A0)were incubated in the in vitro decapping system for the times indicated.7meGDP reaction products were separated by thin layer chromatography andvisualized and quantitated by phosphorimaging.

DETAILED DESCRIPTION OF THE INVENTION

Numerous terms and phrases are used throughout the instantSpecification. The meanings of these terms and phrases are set forthbelow.

In particular, as used herein “half-life” of an RNA molecule refers tothe measurement of the decline in the amount of an RNA molecule.

As used herein “turnover” refers to the degradation of an RNA molecule.Turnover comprises deadenylation and degradation.

As used herein a “cap” or“5′ cap” or “terminal cap”, can be usedinterchangeably, and refer to a 7-methyl guanosine (7meG) cap chemicallyconjugated to the most 5′ nucleotide of the RNA molecule. The 5′-mostphosphate on the RNA will be referred to herein as the “alphaphosphate”.

As used herein, the term “stability” refers to the maintenance of an RNAmolecule so that it can function, and thus retard the degradationprocess of an RNA molecule.

An “RNA element” is a base sequence the possession of which makes anmRNA resist degradation (stability element) or vulnerable to degradation( instability element). Such an element may also be defined as a basesequence to which proteins bind which either contribute to or reducemRNA degradation.

As used herein, the phrase “polyadenylic acid (poly(A)) tail” refers toa string of contiguous adenylic acids (polyadenylate) added posttranscriptionally to the 3′ end of an RNA molecule, such as mRNA.

As used herein, the phrase “a polyadenylic acid competitor nucleic acidoligomer” refers to an oligomer comprising contiguous adenylic acids”which can be added to a system of the invention and sequester proteinsthat bind poly(A). Thus, the degradation of a particular RNA moleculehaving a poly(A) tail can be modulated.

Also, as used herein, the phrase “restriction endonuclease” refers to anenzyme that recognizes specific nucleotide sequences in a nucleic acidmolecule, and produces a double-stranded break within or near the site.Some restriction enzymes, such as EcoRI or HindIII produce“complementary tails” on each of fragments produced. These tails aresaid to be “sticky” because under hybridization conditions they canreanneal with each other. Thus, if two separate nucleic acid moleculesshare the same restriction site, then both will contain complementarysingle-stranded tails when treated with the same restrictionendonuclease, and can be spliced together forming a recombinant nucleicacid molecule.

Naturally, as used herein, the phrase “restriction endonuclease site”refers to a specific nucleotide sequence that is recognized by aspecific restriction endonuclease.

Furthermore, numerous conventional molecular biology, microbiology, andrecombinant DNA techniques within the skill of the art can be readilyutilized to practice the instant invention. Such techniques areexplained fully in the literature. See, e.g., Sambrook, Fritsch &Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. A “replicon” is any genetic element (e.g.,plasmid, chromosome, virus) that functions as an autonomous unit of DNAreplication in vivo, i.e., capable of replication under its own control.

A “cassette” refers to a segment of a nucleic acid molecule, such as DNAor RNA, that can be inserted into a vector at specific restrictionsites. The segment of the nucleic acid molelcule may encode apolypeptide of interest, and the cassette and restriction sites aredesigned to ensure insertion of the cassette in the proper reading framefor transcription and translation.

A cell has been “transfected” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. A cell has been “transformed”by exogenous or heterologous DNA when the transfected DNA effects aphenotypic change. Preferably, the transforming DNA should be integrated(covalently linked) into chromosomal DNA making up the genome of thecell.

A “polynucleotide” or “nucleic acid molecule” refers to the phosphateester polymeric form of ribonucleosides (adenosine, guanosine, uridineor cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine,deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), orany phosphoester analogs thereof, such as phosphorothioates andthioesters, in either single stranded form, or a double-stranded helix.Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. Theterms polynucleotide and nucleic acid molecule, and in particular DNA orRNA molecule, refers only to the primary and secondary structure of themolecule, and does not limit it to any particular tertiary forms. Thus,this term includes double-stranded DNA found, inter alia, in linear orcircular DNA molecules (e.g., restriction fragments), plasmids, andchromosomes. In discussing the structure of particular double-strandedDNA molecules, sequences may be described herein according to the normalconvention of giving only the sequence in the 5′ to 3′ direction alongthe nontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.The boundaries of the coding sequence are determined by a start codon atthe 5′ (amino) terminus and a translation stop codon at the 3′(carboxyl) terminus. A coding sequence can include, but is not limitedto, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNAsequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNAsequences. If the coding sequence is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

The inventors herein have discovered a novel enzyme in cytoplasmicextracts from mammalian cells that specifically removes the 5′ capstructure from mRNAs. Heretofore unrecognized and unappreciated in amammalian system, an in-vitro system has been developed therefrom inwhich this ‘decapping activity’ may be regulated by a variety ofelements in the messenger RNA (RNA elements), including AU-rich elementsand pyrimidine-rich elements, that are known to play a key role indetermining the relative half life of transcripts. Thus, the effects ofthe various components in the system, particularly the target mRNAitself, may be studied in detail using the methods of the invention. Theavailability of the mammalian decapping system of the invention enablesfor the first time several useful activities such as but not limitedto 1) high throughput screening of compounds/macromolecules that affectthe decapping of mammalian mRNAs in order to design drugs to affect theexpression of selected transcripts; 2) purification, characterizationand cloning of proteins and enzymes involved in mRNA decapping, suchproteins and enzymes being potential pharmacologic agents or targets; 3)the development as a diagnostic aid for determining the molecular defectin selective disease alleles; 4) development of in vitro mRNA decappingsystems for other eukaryotic organisms, such as parasites, for which theidentification of differences between the mammalian and non-mammaliandecapping can lead to novel drug discovery; and 5) improving genedelivery systems by using the system of the invention to identifyfactors and RNA sequences that affect RNA stability at the level ofdecapping. These uses are merely exemplary and non-limiting, and will bedescribed in more detail below.

The mammalian in vitro mRNA decapping system comprises the followingbasic components, each of which may be substituted for by likealternates, as described further below:

-   -   1) a mammalian cell cytoplasmic extract;    -   2) a methylated cap analog; and    -   3) a cap-labeled RNA substrate.

The cap-labeled RNA substrate may also contain a poly(A) tail, RNAelements, etc.

The mammalian cytoplasmic extract may be derived, for example, from HeLaS3 cells, but it is not so limited, and may be prepared by clearing ofribosomes and other organelles from a cytoplasmic extract from the cellsusing a standard protocol such as that described by Dignam et al., 1983,Accurate transcription initiation by RNA polymerase II in a solubleextract from isolated mammalian nuclei, Nucleic Acids Res11(5):1475-1489, with the following modifications: (1) extracts areadjusted to 10% glycerol prior to dialysis to maintain the activity ofthe components and (2) dialysis times are reduced to 30 min. for similarreasons. Other ways to maintain stability of the extract are readilyused and fully embraced herein, and the term “cell cytoplasmic extract”is inclusive of the various means of preparing a polysome-free,high-speed supernatant prepared from lysed mammalian cells. An exampleof such an extract is an S100 extract. An extract which is as free ofribosomes as possible within the compass of methods known in the art ispreferred.

The methylated cap analog may be, by way of non-limiting example,^(7me)GpppG or ^(7me)GTP. Any methylated cap analog may be used in thisinvention. Other examples of methylated cap analogs are 7meGpppA,7meGpppC, 7meGpppU, in which the A, C, and U may be unmethylated ormethylated in one or more positions. These are known reagents which maybe synthesized by conventional methods or obtained commercially, forexample, from Amersham Pharmacia Biotech (see also Darzynkiewicz,Nucleosides Nucleotides 18:1125-1126 (1999).

The cap-labeled RNA substrate (which may also contain a poly(A) tail,instability elements, etc) may be any capped mRNA substrate for whichdecapping activity may be measured. In a non-limiting example, the mRNAis labeled at the alpha phosphate of the cap. The label may be aradioactive or non-radioactive isotopic label, or any other detectablegroup which does not interfere with the decapping system, such as butnot limited to a fluorescent moiety (i.e., a fluorigenic substrate), avisibly-detectable moiety (a chromogenic substrate), or a releasablesubstrate or co-factor for a chemical or enzymatic reaction. Theseexamples are merely exemplary and non-limiting, as the assay format maybe designed to be carried out by other manual, semiautomatic orautomatic procedures, the latter useful in particular forhigh-throughput screening.

The foregoing components of the decapping system may be assembled in avariety of ways to achieve the decapping in vitro. Generally, thefollowing components are present, the volumes and concentrationsvariable depending on the particular assay:

-   -   1 microliter of a solution of a methylated cap analog (50-500        ng/microliter);    -   1 microliter cap-labeled RNA (˜5-200 fmol);    -   4 microliters mammalian cell S100 extract; and    -   4 microliters water.

The mixture is incubated at about 30° C. for about 30 min; aliquots arethen taken at designated time points to follow the kinetics of decappingby visualization of the product of decapping, ^(7me)GDP, from thecap-labeled RNA.

Once the parameters of a particular decapping assay are determined, theassay may be simplified and fewer time points measured, or a baselineand single time point may be measured. A simplified assay permits alarge number assays to be performed concurrently.

In one embodiment of a manual procedure, the components are as follows(referred to herein as “standard reaction conditions”):

-   -   1 microliter ^(7me)GpppG or ^(7me)GTP competitor (50-500        ng/microliter);    -   1 microliter RNA (˜5-200 fmol; labeled at the alpha phosphate of        the cap);    -   4 microliters S100 extract; and    -   4 microliters water.

The mixture is incubated at 30° C. for 30 min; aliquots are then takenat designated time points to follow the kinetics of decapping byvisualization of the product of decapping, ^(7me)GDP, by, for example,thin layer chromatography on a cellulose PEI sheet using a mobile phaseof 0.45 M ammonium sulfate. Other means for identifying the decappingproduct are fully embraced herein.

As will be shown in the examples herein and the accompanying figures,the methylated cap analog, ^(7me)GpppG or ^(7me)GTP in the examplesabove, is a key reagent to achieve mRNA decapping in vitro. As seen inthe examples below, standard S100 extracts from human cells exhibit nodecapping. The addition of cold ^(7me)GpppG or ^(7me)GTP, howeveractivates the human cytoplasmic extract and allows one to observedecapping in real time. The titration of methylated cap analogs into thesystem results in the removal of cap binding proteins from 5′ end ofcapped RNA substrates incubated in the system. Furthermore, decapping isshown to be negatively influenced by a poly(A) tail on RNA substrates.mRNAs are not decapped in vivo until they are deadenylated, that isuntil their poly(A) tails are significantly shortened.

A confirmation that the in-vitro decapping system of the invention istruly reproducing in vivo observations is shown by the effect of apoly(A) tail on RNA substrates incubated in the assay of the inventionshould be a strong negative influence on decapping. As seen in theexample, this is precisely the case. Thus, the system of the inventionis accurately reproducing processes that occur inside the cell.

Decapping of RNAs in the system is regulatable by RNA elements. The bestcharacterized mRNA stability element is an AU-rich sequence. Theexistence of AU-rich elements is known in the art. (See, for example,Chen and Shyu (1995); Shyu and Kamen (1986); Shyu, et al. (1991); Shyu,et al. (1989); and Wilson and Treisman (1988)). Proteins which interactwith these elements are also known (see for example Brewer (1991); Fanand Steitz (1998); Lai, et al. (1999); Levine, et al. (1993); Loflin, etal. (1999); Ma, et al. (1996); Peng, et al. (1998). The presence of thiselement in an mRNA in results in a dramatic destabilization of thetranscript in vivo. The addition of such an element, for example anAU-rich element from the TNF-alpha mRNA into the RNA substrates resultsin a dramatic increase in the rate of decapping. The AU-rich elementfrom the GMCSF mRNA also results in a dramatic increase in the rate ofdecapping in the system. The in vitro decapping assay accuratelyreproduces cellular regulatory influences and may thus be used identifythe regulatory mechanisms and learn how to manipulate them intherapeutic interventions. Similarly, a pyrimidine-rich element, forexample such an element which is approximately 50 bases in length, actsas a stability element by significantly decreasing decapping activity.Thus the presence of this element in an mRNA results in stabilization ofthe transcript in vivo. Addition of such an element, for example the 51base pyrimidine-rich element from alpha-globin mRNA, results in adramatic decrease in the rate of decapping, e.g. up to a 10-folddecrease in decapping efficiency (FIG. 9). The existence ofpyrimidine-rich elements is known in the art (see for example ChkheidzeA N, Lyakhov D L, Makeyev A V, Morales J, Kong J, Liebhaber S A.Assembly of the alpha-globin mRNA stability complex reflects binaryinteraction between the pyrimidine-rich 3′ untranslated regiondeterminant and polyC binding protein alphaCP. Mol Cell Biol. 199919:4572-81; Liebhaber SA.mRNA stability and the control of geneexpression. Nucleic Acids Symp Ser. 1997;(36):29-32; Russell J E,Morales J, Makeyev A V, Liebhaber S A. Sequence divergence in the 3′untranslated regions of human zeta- and alpha-globin mRNAs mediates adifference in their stabilities and contributes to efficientalpha-to-zeta gene development switching.Mol Cell Biol. 1998 18:2173-83;Holcik M, Liebhaber S A. Four highly stable eukaryotic mRNAs assemble 3′untranslated region RNA-protein complexes sharing cis and transcomponents. Proc Natl Acad Sci USA. 1997 94:2410-4; Russell J E, MoralesJ, Liebhaber S A.The role of mRNA stability in the control of globingene expression.Prog Nucleic Acid Res Mol Biol. 1997;57:249-87; andWeiss I M, Liebhaber S A.Erythroid cell-specific mRNA stability elementsin the alpha 2-globin 3′ nontranslated region. Mol Cell Biol. 199515:2457-65).

The invention is also directed to kits for carrying out mRNA decappingcomprising at least a mammalian cell cytoplasmic extract and amethylated cap analog. The extract may be prepared in 10% glycerol priorto dialysis, to maintain stability. Optionally included with the kit, orprovided separately, is a detectably-cap-labeled mRNA substrate. Othercomponents and instructions may be included.

The cell extract of the present invention is prepared from lysedmammalian cells or tissues. Various methods known to the skilled artisanmay be used to prepare the cell extract. Various sources of cells may beused, including fresh cells and tissues, and cells lines. Such cells maycomprise foreign nucleic acid, such as in cells that are infected; orare transiently or stably transfected with a mammalian expressionvector, the latter as described in more detail below. Furthermore, priorto preparation of the cell extract, cells may be exposed to certainchemical or other extracellular stimuli, for example, hormones, growthfactors, and kinase and phosphatase inhibitors, which may alter RNAdecapping, for which subsequent studies as described herein may be usedto identify the induction of certain proteins involved in modulating RNAdecapping, or for the identification of agents which may counteractadverse RNA decapping modulation induced by such stimuli. The cellextract is preferably free of nuclei and nuclearcontents and comprisescytoplasm, but this is not essential unless particular components, suchas enzymes or other factors, from nuclei, interfere with the operationof the system. In a typical preparation (for example an S100 extract),which may be modified without departing from the scope of the invention,cells are grown, harvested, lysed, centrifuged for 100,000×g for 1 hour,and dialyzed. Glycerol may be added to protect the extract if storedfrozen. Variations in the preparation of a 100,000×g mammalian cellsupernatant may be undertaken which provides a suitable component forthe system of the invention, and such variations are fully embracedherein (for example an extract centrifuged at a different rate or time,such as 99,000×g, etc.).

Cells useful for the preparation described herein include immortalizedor partially immortalized mammalian cells which can be grown in largeamounts under defined conditions, such as HeLa cells and various T-cellcell lines. Other sources include tissues, blood cells, or myeloidcells. Other sources are well within the realm of the present invention.

Also part of this invention is a polypeptide with the following uniquecombination of properties: a molecular weight of about 50 to about 100kilodaltons (kD) in molecular exclusion chromatography; precipitation by20% ammonium sulfate, elution at between about 440 to 500 mM NaCl from aheparin-Sepharose column, and the ability to decap mammalian RNA. In oneembodiment, this polypeptide causes decapping of a cap-labelled mRNAsubstrate when combined with a methylated cap analog. This polypeptideis therefore called a “decapping enzyme”. The decapping enzyme may beused in various ways suggested by this invention. For example, theenzyme may be used to target specific mRNAs for degradation by attachingto the enzyme a nucleic acid sequence which hybridizes to the mRNA. Whenhybridization occurs, the enzyme will then act to decap the mRNA,leading directly to its degradation. This enzyme targeting system isparticularly useful for degrading mRNAs which translate into defectiveor otherwise undesirable proteins, or simply to regulate the levels ofcertain proteins. As is apparent from this discussion, such a systemalso is useful as a research tool for drug discovery. An addition, thedecapping enzyme may itself be used to probe cell extracts for otherfactors which bind to it in nature and therefore regulate its activity,providing additional guidance on targeting the decapping enzyme tospecific mRNAs.

The polypeptide of this invention may be obtained by known methods, suchas chemical synthesis, or by recombinant DNA techniques, a recombinantDNA technique being one in which a polynucleotide coding for saidpolypeptide is added to a host cell so as to result in the expression ofthe polypeptide. The polypeptide can be purified away from host cellproteins by such methods as immunoprecipitation with antibodies and byother standard protein purification techniques such as electrophoresisin one or two dimensions and chromatography. The polypeptide of thisinvention may also be isolated from tissue or cells which naturallyexpress it, or from cultured cells which express it, such as HeLa cells.Such isolation employs known methods of isolating proteins followed byassays and purification steps as described below to obtain thepolypeptide of this invention with its defining characteristics.

A polynucleotide which encodes the polypeptide of this invention is alsocontemplated. Such a polynucleotide, when expressed in a conventionalexpression system as described below, expresses a polypeptide with thefollowing unique combination of properties: a molecular weight of about50 to about 100 kilodaltons (kD) in molecular exclusion chromatography;precipitation by 20% ammonium sulfate, elution at between about 440 to500 mM NaCI from a heparin-Sepharose column, and the ability to decapmammalian RNA. The polynucleotide of this invention may be used, in oneaspect, to obtain the polypeptide of this invention.

Monoclonal and polyclonal antibodies which bind to the polypeptide ofthis invention. preferably with high affinity and specificity, are alsocontemplated. Such antibodies are raised by known methods, such ashybridoma technology in the case of monoclonal antibodies, andcollection from an immunized laboratory animal in the case of polyclonalantibodies. Thus an antibody which binds specifically and with highaffinity to a polypeptide having a molecular weight of about 50 to about100 kilodaltons (kD) in molecular exclusion chromatography,precipitating with 20% ammonium sulfate, eluting at between about 440 to500 mM NaCl from a heparin-Sepharose column, and the able to decapmammalian RNA is part of this invention. By binding specifically ismeant the conventional meaning of binding to the polypeptide of thisinvention and showing no significant binding to other antigens. Highaffinity is also used here in the sense that it is generally understoodin the art. The antibodies of this invention may be used as researchtools in drug discovery, and in addition may be used for example toprevent mRNA degradation by blocking the action of the decapping enzyme.The antibodies may also be used analytically and diagnostically, forexample to determine levels of decapping enzyme in a particular cell ortissue.

As described above, a cell used to prepare the cell extract may compriseforeign DNA, and can be prepared as described below. A polynucleotide ofthis invention can be obtained and/or expressed using a large number ofvector-host systems known in the art. Possible vectors include, but arenot limited to, plasmids or modified viruses, but the vector system mustbe compatible with the host cell used. Examples of vectors include, butare not limited to, E. coli, bacteriophages such as lambda derivatives,or plasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g.,pGEX vectors, pmal-c, pFLAG, etc. The insertion into a cloning vectorcan, for example, be accomplished by ligating the polynucleotide into acloning vector which has complementary cohesive termini. However, if thecomplementary restriction sites used to fragment the polynucleotide arenot present in the cloning vector, the ends of the molecule may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the termini of thepolynucleotide; these ligated linkers may comprise specific chemicallysynthesized oligonucleotides encoding restriction endonucleaserecognition sequences. Recombinant molecules can be introduced into hostcells via transformation, transfection, infection, electroporation,etc., so that many copies of the polynucleotide are generated.Preferably, the cloned polynucleotide is contained on a shuttle vectorplasmid, which provides for expansion in a cloning cell, e.g., E. coli,and facile purification for subsequent insertion into an appropriateexpression cell line, if such is desired. For example, a shuttle vector,which is a vector that can replicate in more than one type of organism,can be prepared for replication in both E. coli and Saccharomycescerevisiae by linking sequences from an E. coli plasmid with sequencesfrom the yeast 2μ plasmid.

Naturally, any of the methods previously described for the insertion ofan isolated polynucleotide into a cloning vector may be used toconstruct expression vectors containing a polynucleotide consisting ofappropriate transcriptional/translational control signals and theprotein coding sequences. These methods may include in vitro recombinantDNA and synthetic techniques and in vivo recombination (geneticrecombination).

Mammalian expression vectors contemplated for use in the inventioninclude vectors with inducible promoters, such as the dihydrofolatereductase (DHFR) promoter, e.g., any expression vector with a DHFRexpression vector, or a DHFR/methotrexate co-amplification vector, suchas pED (PstI, SalI, SbaI, SmaI, and EcoRI cloning site, with the vectorexpressing both the cloned gene and DHFR; see Kaufman, Current Protocolsin Molecular Biology, 16.12 (1991). Alternatively, a glutaminesynthetase/methionine sulfoximine co-amplification vector, such as pEE14(HindIII, XbaI, SmaI, SbaI, EcoRI, and BclI cloning site, in which thevector expresses glutamine synthase and the cloned gene; Celltech). Inanother embodiment, a vector that directs episomal expression undercontrol of Epstein Barr Virus (EBV) can be used, such as pREP4 (BamH1,SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning site,constitutive RSV-LTR promoter, hygromycin selectable marker;Invitrogen), pCEP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII,and KpnI cloning site, constitutive hCMV immediate early gene,hygromycin selectable marker; Invitrogen), pMEP4 (KpnI, PvuI, NheI,HindIII, NotI, XhoI, SfiI, BamH1 cloning site, inducible metallothioneinIIa gene promoter, hygromycin selectable marker: Invitrogen), pREP8(BamH1, XhoI, NotI, HindIII, NheI, and KpnI cloning site, RSV-LTRpromoter, histidinol selectable marker; Invitrogen), pREP9 (KpnI, NheI,HindIII, NotI, XhoI, SfiI, and BamH1 cloning site, RSV-LTR promoter,G418 selectable marker; Invitrogen), and pEBVHis (RSV-LTR promoter,hygromycin selectable marker, N-terminal peptide purifiable via ProBondresin and cleaved by enterokinase; Invitrogen). Selectable mammalianexpression vectors for use in the invention include pRc/CMV (HindIII,BstXI, NotI, SbaI, and ApaI cloning site, G418 selection; Invitrogen),pRc/RSV (HindIII, SpeI, BstX, NotI, XbaI cloning site, G418 selection;Invitrogen), and others. Vaccinia virus mammalian expression vectors(see, Kaufman, 1991, supra) for use according to the invention includebut are not limited to pSC11 (SmaI cloning site, TK- and β-galselection), pMJ601 (SalI, SmaI, AflI, NarI, BspMII, BamHI, ApaI, NheI,SacII, KpnI, and HindIII cloning site; TK- and β-gal selection), andpTKgptF1S (EcoRI, PstI, SalI, AccI, HindII, SbaI, BamHI, and Hpa cloningsite, TK or XPRT selection).

Once a particular polynucleotide is inserted into a vector, severalmethods known in the art may be used to propagate it. Once a suitablehost system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity. Aspreviously explained, the expression vectors which can be used include,but are not limited to, the following vectors or their derivatives:human or animal viruses such as vaccinia virus or adenovirus; insectviruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g.,lambda), and plasmid and cosmid DNA vectors, to name but a few. Inaddition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired to express the polypeptide ofthis invention.

Vectors are introduced into the desired host cells by methods known inthe art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun or a DNAvector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem.267:963-967; Wu and Wu, 1988. J. Biol. Chem. 263:14621-14624; Hartmut etal., Canadian Patent Application Ser. No. 2,012,311, filed Mar. 15,1990).

The target, cap-labeled mRNA sequence in the system of the presentinvention may be any one of a number of RNA or modified RNA molecules.For example, synthetic RNA may be prepared by solid phase synthesis, orreproduced by in-vitro transcription using phage polymerase as is knownto the skilled artisan. Naturally occurring RNA may be isolated fromcells, tissues, and other biological sources. Cap-labelling may beachieved by methods known in the art.

The particular mRNA used in the system and methods of the presentinvention may be selected depending on the particular species of mRNA tobe studied. Investigations of mRNA turnover, endogenous modulators ofits turnover and exogenously added molecules, particularly smallmolecules which affect mRNA turnover, have important therapeuticimplications in the prophylaxis and treatment of a variety of conditionsand diseases. Certain mRNAs are short-lived, such as those of cytokines;others are long-lived, such as globin message. The regulation of mRNAlifetimes for particular proteins and particular cell types may besubject to various adverse effects, from infection to external stimuli,which alter the turnover and hence cellular physiology. In variousconditions, altered expression of cellular proteins and cellularphenotypes may be consequences of altered mRNA turnover. Pharmacologicalintervention of such altered mRNA turnover, to restore an alteredturnover, or the induction of an altered turnover to achieve a benefitto the organism, are achievable based upon the systems and methodsdescribed herein. For example, a particular mRNA, such as that of theproinflammatory cytokine TNF-alpha, is selected as a target foridentification of small molecule modulators that may decrease theturnover by affecting decapping, and this prolong the lifetime, andexpression, of this protein by inflammatory cells. Such modulators mayprovide substantial benefit in the treatment of certain immunologicaldiseases wherein an increased secretion of TNF-alpha is beneficial.Conversely, massive overproduction of TNF-alpha in sepsis, or itsadverse effects in rheumatoid arthritis and inflammatory bowel diseasemay be ameliorated by use of an agent which further increases theturnover and thus decreases the expression of TNF-alpha by inflammatorycells.

The application of the invention herein to other mRNA species isembraced by the teachings herein. In particular, the methods of thepresent invention facilitate high throughput screening for theidentification of modulators of RNA decapping, to be applied to thetreatment or prophylaxis of disease.

Furthermore, the cap-labeled mRNA molecule may be detectably labeledusing routine protocols readily known to a skilled artisan. Suitablelabels include enzymes, fluorophores (e.g., fluorescein isothiocyanate(FITC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chelatedlanthanide series salts, especially Eu³⁺, to name a few fluorophores),chromophores, radioisotopes, chelating agents, dyes, colloidal gold,latex particles, ligands (e.g., biotin), and chemiluminescent agents.Capped RNAs are preferably labelled with alpha-³²p GTP. When a controlmarker is employed, the same or different labels may be used for thereceptor and control marker. In the instance where a radioactive label,such as the isotopes ³H, ¹⁴C, ³²p, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe,⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re are used, known currently available countingprocedures may be utilized. In the instance where the label is anenzyme, detection may be accomplished by any of the presently utilizedcolorimetric, spectrophotometric, fluorospectrophotometric, amperometricor gasometric techniques known in the art. In a further example, biotinmoieties may be incorporated into the RNA cap by any number of means.Subsequently, the biotinylated RNA or degradation fragments may bequantitated by an avidin reagent.

Direct labels are one example of labels which can be used according tothe present invention. A direct label has been defined as an entity,which in its natural state, is readily visible, either to the naked eye,or with the aid of an optical filter and/or applied stimulation, e.g.U.V. light to promote fluorescence. Among examples of colored labels,which can be used according to the present invention, include metallicsol particles, for example, gold sol particles such as those describedby Leuvering (U.S. Patent 4,313,734); dye sole particles such asdescribed by Gribnau et al. (U.S. Pat. No. 4,373,932) and May et al. (WO88/08534); dyed latex such as described by May, supra, Snyder (EP-A 0280 559 and 0 281 327); or dyes encapsulated in liposomes as describedby Campbell et al. (U.S. Pat. No. 4,703,017). Other direct labelsinclude a radionucleotide, a fluorescent moiety or a luminescent moiety.In addition to these direct labelling devices, indirect labelscomprising enzymes can also be used according to the present invention.Various types of enzyme linked immunoassays are well known in the art,for example, alkaline phosphatase and horseradish peroxidase, lysozyme,glucose-6-phosphate dehydrogenase, lactate dehydrogenase, urease, theseand others have been discussed in detail by Eva Engvall in EnzymeImmunoassay ELISA and EMIT in Methods in Enzymology, 70. 419-439, 1980and in U.S. Pat. No. 4,857,453.

Thus, the assay of the invention which achieves mammalian mRNA decappingin vitro is useful for a variety of studies, such as but not limited toidentifying the components involved in decapping, identifying cells ortissues with defects in decapping and possible pharmaceutical agentsthat can restore activity, the identification of inhibitors ofdecapping, screening of molecules that modulate decapping activity, toname but a few examples. The decapping assay of the invention can becombined with reagents used to measure mRNA stability, such asdeadenylation and/or degradation, in order provide a system in which theentire process of mRNA turnover is assessed in a single container. Thus,the compositions and kits described above may also include reagentsforcarrying out additional aspects of RNA turnover.

Thus, a method is also provided for identifying an agent capable ofmodulating the decapping of a target mRNA sequence comprising

-   -   (A) preparing the decapping system of the present invention;    -   (B) introducing said agent into said system; and    -   (C) correlating any effect of the agent on decapping with        modulation of the decapping of said target RNA sequence in said        system.

A further method is provided for identifying a defect in a mammaliancell cytoplasmic extract (such as an S100 extract) responsible foraltered decapping activity in a certain cell or tissue comprising:

-   -   (A) preparing the system of the present invention using a        mammalian cell cytoplasmic extract (such as an S100 extract)        from a particular cell or tissue; and    -   (B) monitoring the extent of decapping of said target RNA        sequence in said system.

The foregoing method is useful in identifying a defect present in a cellor tissue. Components may be added to the foregoing system to attempt tocircumvent the block in normal decapping activity.

The inventors herein have identified and characterized a novel regulateddecapping activity in mammalian cytoplasmic extracts which plays a keyrole in mRNA turnover. Decapping was found to be repressed by twoactivities: cap binding proteins and poly(A) binding proteins. Decappingwas found to be directly stimulated by AU-rich elements, and directlyinhibited by pyrimidine-rich elements. The compositions and systems ofthe invention may include such decapping activity modulators as baselinecomponents or the target mRNA from which to study or identify modulatorsof decapping as altered by the presence of these elements.

The present invention may be better understood by reference to thefollowing non-limiting Examples, which are provided as exemplary of theinvention. The following examples are presented in order to more fullyillustrate the preferred embodiments of the invention. They should in noway be construed, however, as limiting the broad scope of the invention.

EXAMPLES

A. Materials and Methods for Performing Experimental Procedures

1. RNAs

SVARE-A0 RNA, which contains the 34 base AU-rich element from TNF-alphainserted into the BamH I-Bcl I fragment representing the 3′ portion ofSV40 late mRNAs, was transcribed from Hind III linearized templates aspreviously described (Ford et al., 1999). SVARE-A60 RNA, a variant thatcontains a 60 base poly(A) tract at its 3′ end, was prepared aspreviously described (Ford et al., 1999). Gem-A60 RNA, which containssequences from the pGem4 polylinker region followed by 60 A residues,was prepared as previously described (Ford et al., 1999). GemARE-A60, avariant that contains the 34 base AU-rich element from TNF-alpha, wasprepared as described previously (Gao et al., 2000). Transcription ofHind III linearized templates yields GemARE-A0 RNA. Sequences encoding a60 base poly(A) tail were added to DNA using a ligation-PCR approachpreviously described (Ford et al., 1997). The template for GM-CSF andGMCSF (-ARE) was pGM-CSF (Shaw and Kamen, 1986) cut with EcoR I (toyield a 750 base transcript) or Nco I (to yield a 515 base transcript)respectively. GMCSFT-A0 RNA was prepared by inserting the AU-richelement from TNF-alpha into pGMCSF by inserting the oligonucleotide5′-CATGATTATTTATTATTTATTTATTATTTATTTATTTAAAC (SEQ ID NO: 1) and itsappropriate complement at its Nco I site. This replaced the endogenousdestabilizing element of GMCSF with the TNF-alpha AU-rich element.Transcription of Nco I linearized templates yields a 557 base GM-CSFT-A0RNA. The addition of a 60 base poly(A) tail to GM-CSFT A0 RNA wasperformed by ligation-PCR as described above.

RNAs were transcribed in vitro using SP6 polymerase as describedpreviously (Wilusz and Shenk, 1988) in the absence of cap analog andradioactive rNTPs. To label RNAs exclusively at their cap structures,transcription products were then capped using recombinant vacciniaguanyltransferase and alpha-³²P-GTP (Zhang et al., l999b). All RNAs werepurified on 5% acrylamide gels prior to use. To make RNAs that wereradioactively labeled at the alpha-phosphate of an unmethylated cap,S-adenosyl homocysteine was substituted for S-adenosyl methionine in thecapping reaction.

Synthetic RNAs used in competition studies were made by the NJMSMolecular Core Facility and contained the following sequences: ARE:5′AUUAUUUAUUAUUUAUUUAUUAUUUAUUUAUUUA SEQ ID NO: 2); Non-specificcompetitor: 5′-GGAUUAACUAAUUGAUACCGCGUAUACACGCGG (SEQ ID NO:3). Poly(A)and poly(C) competitor RNAs, along with ^(7me)GpppG and GpppG werepurchased from Amersham Pharmacia Biotech.

2. Extracts

Whole-cell yeast extracts were prepared as described (Lin et al., 1985).S100 cytoplasmic extracts were prepared from HeLa spinner cells grown in10% horse serum as described previously (Ford et al., 1999; Ford andWilusz, 1999). Aliquots were stored at −80° C.

3. In vitro Decapping Assay

Decapping assays were performed using conditions adapted from Zhang etal (1999b). 10-50 fmoles of cap-labeled RNAs were incubated with 4microliters HeLa S100 cytoplasmic extract in a 10 microliter reaction inthe presence of CE buffer (50 mM Tris pH 7.9, 30 mM (NH₄)₂SO₄, 1 mMMgCl₂) and 20 micromolar cap analog (where indicated). Reaction mixtureswere incubated at 30° C. for the times indicated and stopped by theaddition of 1 microliter of 0.25 M EDTA. Reaction products wereseparated and identified by thin layer chromatography on PEI cellulosesheets developed in 450 mM (NH₄)₂SO₄. Quantitation was performed using aMolecular Dynamics Phosphorimager. Twenty micrograms of ^(7me)CGMP and^(7me)GDP were routinely spotted on TLC plates along with reactionsamples to serve as markers that could be visualized by UV shadowing.

4. UV Cross-Linking

UV cross-linking analysis was performed as described (Wilusz and Shenk,1988). Briefly, 20-50 fmoles of cap-radiolabeled RNA was incubated inthe in vitro decapping assay for 5 min. and then reaction mixtures wereirradiated on ice for 10 min. using a 15W germicidal light. RNases A andT1 were added and proteins covalently attached to short radioactive RNAoligomers were analyzed on a 10% acrylamide gel containing SDS. Foranalysis of UV cross-linked DAN/PARN protein by immunoprecipitationusing a polyclonal antibody obtained from M. Wormington (Korner et al.,1998), 400 ul of NET2 buffer (50 mM Tris pH 7.6, 150 mM NaCl, 0.01%NP40) was added to reaction mixtures following RNase treatment andreaction mixtures were centrifuged for 4 min. Pre-cleared samples wereincubated on ice with 2-5 microliters of specified rabbit polyclonalantisera for 1 hr. Antigen antibody complexes were collected on proteinA-positive Staphylococcus aureus cells, washed five times in RIPA buffer(50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% SDS, 1% NP40, 0.5% deoxycholate),and immunoprecipitated cross-linked proteins were analyzed on a 10 or15% acrylamide gel containing SDS.

B. Experimental Procedures and Results

1. Identification of a Decapping Activity in HeLa Cytoplasmic Extracts

Following deadenylation, most yeast mRNAs are decapped prior todegradation by the 5′-to-3′ exonuclease Xm1p (Muhlrad et al., 1994).Whether decapping plays a major role in the turnover of mammalian mRNAsfollowing poly(A) tail shortening is unclear. No direct biochemicalevidence exists for a bona-fide Dcp1p-like decapping enzyme in mammaliancells. In order to obtain a decapping activity in mammalian extracts,the decapping assay was used to study the yeast enzyme (Zhang et al.,1999b). Briefly, RNA substrates were exclusively labeled at thealpha-phosphate of the cap structure using recombinant vaccinia cappingenzyme and alpha-³²P-GTP and incubated in cytoplasmic extracts in thepresence of Mg²⁺. Reaction products were spotted directly onto PEIcellulose sheets and small molecules were resolved in 450 mM ammoniumsulfate. The positions of ^(7me)GMP, ^(7me)GDP and ^(7me)GTP wereidentified by UV shadowing of markers run in each lane. As seen in FIG.1A, yeast whole cell extracts contain a potent decapping activity thatproduces a significant amount of ^(7me)GDP from cap-labeled RNAsubstrates. The ^(7me)GDP product of decapping, however, was notdetected when cap-labeled RNAs were incubated in HeLa S100 extracts. Inaddition, no significant decrease in radioactivity at the origin wasobserved in these experiments. Thus the decapping activity of HeLa cellsis somehow masked in the in vitro assay.

The cap structure of mRNA substrates becomes inaccessible when thetranscript is incubated in HeLa extracts due to competing cap-bindingactivities such as eIF4E (Sonenberg et al., 1978) or the deadenylaseDAN/PARN (Dehlin et al., 2000; Gao et al., 2000). Parker and colleagueshave previously shown that the yeast decapping enzyme Dcp1p is notinhibited by small cap analogs (LaGrandeur and Parker, 1998). Theaddition of ^(7me)GpppG cap analog to extracts selectively sequestersthese competing cap-binding activities without inhibiting the mammaliandecapping enzyme. A decapping activity in HeLa extracts was found byadding 20 micromolar ^(7me)GpppG to reaction mixtures and repeating thedecapping assay using 3 independently prepared HeLa cytoplasmicextracts. As seen in FIG. 1B, the addition of cap analog activateddecapping of RNA substrates in all of the extracts tested as seen by theaccumulation of ^(7me)GDP. The activation of decapping on all RNAsubstrates we have tested required the addition of cap analog.Furthermore, decapping occurred with linear kinetics, was dependent onthe presence of Mg²⁺, and did not require ATP (FIGS. 3 and 5). Finally,the decapping activity was highly specific for ^(7me)Gppp capped RNAs,as unmethylated cap structures were not detectably cleaved in the assays(FIG. 1C). Taken together, these data show that HeLa cytoplasmicextracts contain a decapping activity that is repressed by cap bindingproteins which prevent its access to the cap of RNA substrates.

In order to test the specificity of the activation of decapping in HeLaextracts by cap analog, the ability of methylated versus non-methylatedcap analogs to activate decapping were compared. As seen in FIG. 2A, thepresence of the methyl group on the cap analog was absolutely requiredto activate decapping. Decapping was activated by cap analog through thespecific sequestration of cap-binding proteins. In order to confirmthis, UV cross-linking assays were performed using cap labeled RNAsubstrates. As seen in FIG. 2B, the addition of increasing amounts ofmethylated cap analog to HeLa S100 extracts specifically competed for a24 kDa protein. The 24 kDa protein is likely to be eIF4E (Altmann etal., 1985) since it has a similar apparent molecular weight,specifically cross-links to ^(7me)cap structures, and is only detectedin cytoplasmic extracts. Interestingly, Schwartz and Parker (2000) haverecently demonstrated that purified yeast eIF4E can inhibit purifiedyeast decapping enzyme in vitro. Similar, but not identical amounts of^(7me)GpppG also specifically competed for cap binding by thedeadenylase DAN/PARN (FIG. 2C). In order to determine whether theactivation of decapping was correlated with the competition of the 24kDa cap binding protein and/or DAN/PARN, careful titration experimentswith cap analog were performed. As seen in FIG. 2D, decapping was onlypartially activated by the addition of 5 micromolar cap analog. Thisamount of cap analog fully sequestered the 24 kDa cap binding protein,but not DAN/PARN in the extracts. Full activation of decapping required10micromolar cap analog, the amount required to fully sequesterDAN/PARN. These data show that DAN/PARN may be a key factor inpreventing decapping through interaction with the 5′ cap. Specificremoval of DAN/PARN from extracts by immunodepletion, however, failed toactivate decapping . We conclude that a decapping activity withproperties similar to the yeast Dcp1p enzyme exist in mammaliancytoplasmic extracts and can be specifically activated by sequesteringcap binding activities. These data show that access to the cap structureis an important feature in the regulation of decapping in mammaliancells.

2. A Poly(A) Tail Represses Decapping of Mammalian mRNAs in a Poly(A)Binding Protein-Dependent Fashion

Poly(A) tail shortening is a prerequisite for the decapping and turnoverof most yeast mRNAs (Decker and Parker, 1993). This suggests that thepresence of a poly(A) tail on RNA substrates represses decapping invivo. In order to confirm that the in vitro decapping assay faithfullyreproduces this regulatory aspect of in vivo decapping, matched RNAsubstrates were added to HeLa S100 extracts that either lacked a poly(A)tail (GemARE-A0, SVARE-A0 or GMCSFT-A0) or contained 60 adenylateresidues at their 3′ ends (GemARE-A60, SVARE-A60 or GMCSFT-A60). Thesizes of these transcripts vary from 95 to >700 bases to insure thatresults obtained were independent of the size of the transcript andcould be generalized. As seen in FIG. 3, all 3 transcripts that lacked apoly(A) tail at their 3′ end were efficiently decapped in S100 extractin the presence of cap analog. Their adenylated counterparts, however,were decapped at a dramatically reduced efficiency (10-20 fold). Notethat the position of the ^(7me)GDP spot varies depending on the time ofincubation in the extract. In all cases, however, that radioactive spotco-migrates with ^(7me)GDP as determined by UV shadowing of unlabeledmarkers that were loaded in each lane (altered migration is due to thegeneration of small molecules during the incubation of the extract thatcaused alteration in the chromatographic mobility of nucleotides on PEIcellulose). The presence of a poly(A) tail inhibits decapping in HeLacytoplasmic extracts in a similar manner as has been observed in yeast(Muhlrad and Parker, 1994). The in vitro decapping assay reproduces thisimportant regulatory aspect of mRNA turnover.

In order to assess the involvement of poly(A) binding proteins in themechanism of poly(A) tail-mediated repression of decapping, increasingamounts of poly(A) competitor RNA were added to in vitro decappingassays using an RNA substrate that possessed a 60 base poly(A) tail. Asseen in FIG. 4A, the addition of cold poly(A) competitor RNA thatinhibited the cross-linking of a 70 kDA poly(A) binding protein to thepoly(A) tail (Ford and Wilusz, 1999, data not shown) effectivelystimulated decapping to levels observed with deadenylated substrates.Similar data were obtained for all RNA substrates tested. Since thedecapping assays are performed in the presence of cap analog thatsequesters the deadenylase DAN/PARN, deadenylation of the RNA substrateis not occurring under these conditions (Gao et al., 2000) and,therefore, cannot account for the results obtained. Furthermore, poly(A)competitor RNA failed to stimulate decapping in the absence of capanalog. The presence of free cap-binding proteins in extracts is adominant inhibitor to in vitro decapping. The stimulation of decappingwas specific for poly(A), as other homopolymers such as poly(C) did notappreciably affect decapping of all RNA substrates tested (FIG. 4B).Finally, we tested whether the stimulation of decapping by poly(A)competitor RNA was a general activator of decapping or was specific foradenylated transcripts. As seen in FIG. 4C, the decapping of an RNAsubstrate that lacked a poly(A) tail (GemARE-A0) was only mildlystimulated by the addition of poly(A) competitor RNA. The small(1.5-fold) effect of poly(A) on decapping of the GemARE-A0 RNA substrateis likely due to the low affinity of poly(A) binding protein for theAU-rich element contained in the 3′ portion of the transcript (D. Fritzand J. Wilusz, data not shown). Similar data was also obtained for allRNA substrates tested that lacked a 3′ poly(A) tail. Poly(A) bindingproteins are repressors of decapping when bound to poly(A)⁺ RNAsubstrates. Furthermore, since the decapping assays are performed in thepresence of ^(7me)GpppG that sequesters eIF4E (FIG. 2), the repressionof decapping by poly(A) binding proteins occurs in a novel,eIF4E-independent fashion.

3. AU-Rich Instability Elements Dramatically Stimulate Decapping

Many short-lived mammalian mRNAs contain AU-rich elements in their 3′untranslated regions that have been shown to be directly responsible forthe high rate of turnover of these transcripts (Chen and Shyu, 1995).While AU-rich instability elements have been shown to increase the rateof deadenylation both in vivo (Shaw and Kamen, 1986; Shyu et al., 1989)and in vitro (Ford et al., 1999), efficient deadenylation requires aninteraction between DAN/PARN and the 5′ cap structure (Gao et al.,2000). AU-rich elements are be capable of stimulating deadenylation bymaking the cap structure more accessible to the deadenylase, and AU-richelements are also be able to stimulate decapping by a similar mechanism.Two independent RNA substrates either lacked (Gem-A0 and SV-A0) orcontained (GemARE-A0 and SVARE-A0) the AU-rich element from theTNF-alpha mRNA. As seen in FIGS. 5A and B, the presence of the TNF-alphaAU-rich element in an RNA substrate stimulated decapping over 10-fold.In order to generalize this observation to other AU-rich elements, anRNA that contained the GM-CSF ARE or a matched control transcript thatlacked the element was prepared. As seen in FIG. 5C, the AU-rich elementfrom the GM-CSF mRNA also strongly stimulated decapping in vitro. Weconclude that AU-rich instability elements dramatically stimulatedecapping.

The AU-rich element binding proteins (ARE-BPs) play a role in thestimulation of decapping by the instability element. In order tosequester ARE-BPs in S100 extracts, increasing amounts of a 34 basesynthetic RNA oligomer containing the TNF-alpha AU-rich element (ARE)were added to in vitro decapping assays. In control reactions, similaramounts of an unrelated 33 base RNA were added. As seen in FIG. 6, theaddition of the ARE competitor RNA significantly reduced decapping of anAU-rich element-containing RNA substrate while similar levels of thecontrol competitor RNA had no effect on decapping efficiency. Thereduction in decapping efficiency by the ARE synthetic RNA competitorwas only observed with RNA substrates that contained an AU-rich element(FIG. 6B). Finally, UV cross-linking assays correlated the competitionof a specific ARE-binding protein by the ARE competitor RNA withrepression of decapping. Both ARE-specific RNA binding proteins that weobserved in our UV cross-linking assays, HuR (a known mRNA stabilizer)and an unidentified 40 kDa species (Ford et al., 1999), were competed bythe ARE RNA oligomer with exactly the same concentration dependence andkinetics.

4. Identification and Purification of Human Decapping Protein

The decapping protein (preferably an enzyme) from HeLa cells is purifiedusing a combination of conventional and affinity chromatography steps.The majority of HeLa decapping activity can be precipitated by 20%ammonium sulfate. Molecular exclusion chromatography using a Sepharose-6column indicates that the decapping activity elutes in the ˜50-100 kDarange, consistent with a single (or few) polypeptides being responsiblefor enzymatic activity. Therefore following decapping activity throughpurification will likely not require reconstitution of multiplefractions as would be the case with large multi-component complexes(i.e. 20). The bulk of decapping activity elutes between 440 and 500 mMNaCl from a heparin-sepharose column (see FIG. 8, Panels A, B, C).

a. Fractionation

i. Starting material: S100 cytoplasmic extract from HeLa spinner cellsis prepared in bulk by known methods and as described herein (see, forexample, Ford, et al, 1999 and Ford and Wilusz, 1999).

ii. Assays for factors during fractionation: As the purificationproceeds, decapping activity is followed through sequential purificationsteps using the In vitro Decapping Assay described above in section A.3.This assay is easy to perform, gives rapid and quantitative results, andis highly reproducible. Since the yeast decapping enzyme is contained ina single polypeptide, individual fractions are first tested to identifyHeLa decapping activity, and appropriate fractions mixed/matched ifnecessary to reconstitute active decapping. Fraction purity is assessedby silver staining of fractions run on SDS-PAGE gels using knownmethods.

iii. Purification:. Extracts are initially fractionated by an ammoniumsulfate cut of 20% saturation. All of the decapping activity isprecipitated at these levels of saturation. A range of conventional andwell known fractionation methods on an analytical scale may be used foroptimal purification protocols. At each step, fractions are assayedusing the decapping assay in order to confirm that the decapping protein(or enzyme) is present in the fraction.

The available methods include anion exchange columns (DEAE),phosphocellulose, cation exchange columns (S-sepharose, heparin),molecular exclusion (Superose 6), hydrophobic interactions columns(phenyl-sepharose) and step elutions to identify optimal purificationsteps. Affinity columns such as dye ligand chromatography and nucleicacid columns are also available. Linear elution gradients are employedto optimize purification efficiencies. As purification continues,fractions may be mixed and matched as required to reconstitute activity.

Successful analytical steps may be increased to preparative scale usingavailable high resolution columns (Amersham-Pharmacia). Buffer exchangeswill be performed using conventional dialysis or Centricon columns whereapplicable. An automated Amersham-Pharmacia AKTA purification system, UVmonitor and fraction collector may used for these efforts. Allpurifications are preferably performed at 4 degrees C. in the presenceof protease inhibitors (PMSF, leupeptin and others, if necessary).

Fractions should be kept as concentrated as possible, and glycerol andDTT should be present in all buffers, and NP40 or carrier proteins maybe added to further maintain stability if necessary. Fractions are bestmaintained if quick frozen in liquid nitrogen and stored at −80 degreesC.

b. Molecular Characterization of the Mammalian Decapping Protein

i. Peptide sequencing: Following purification to near homogeneity,proteins that co-fractionate with decapping activity are excised fromdenaturing gels and subjected to mass spectrophotometry and/ormicro-sequencing by known methods. Since many peptide sequences are nowknown, MALDI-MS should be performed on samples as a first step followedby database searches using Peptide Search I software.

ii. cDNA clones: Identified cDNA clones may be obtained by manytechniques, most conveniently by RT-PCR amplification using appropriateprimers based on the above sequence analysis. Alternatively, libraryscreening can be performed by conventional methods using frozen HeLacDNA libraries using appropriate probes based on the above sequenceanalysis.

iii. Full-lenoth clones: Full-length clones of the decapping activityare obtained as described above and may be sequenced and analyzed forinteresting motifs that will yield insights into its structure/functionand/or regulation. Such motifs include enzymatic signatures, RNA bindingdomains, phosphorylation sites, etc. Full-length ORFs are cloned intobacterial expression vectors (such as pGEX and/or pRSET) and induced toexpress high levels of recombinant decapping protein(s). Alternatively,yeast or baculovirus expression systems can be used if proteins made inbacteria are difficult to express or are inactive. Proteins will bepurified using Ni or glutathione affinity columns and tested fordecapping activity in vitro with cap-labeled RNA substrates to confirmthe identity of the decapping enzyme. Alternatively, HeLa cell proteinseluted from denaturing gels can be renatured (100) to identify enzymaticsubunits.

iv. Antibodies: Antibodies may be generated using conventional methodsfrom the isolated and purified peptide(s) having decapping activity.These immunologic reagents will be very useful in assessing (A) thesubcellular localization (an especially interesting question given thenuclear association of some aspects of nonsense-mediated decay processthat may be regulated through decapping); (B) developmental andtissue-specific expression patterns; (C) assessing post-translationalmodifications of decapping proteins; (D) depletion/add back studies; and(E) as an invaluable reagent for future protein-protein interactionstudies to identify factors that associate with the decapping enzyme andmay regulate its activity. Both monoclonal and polyclonal antibodies arecontemplated.

5. Effect of Pyrimidine-Rich Stability Element on Decapping Rates

The alpha-globin element was inserted into three independent RNAsubstrates and in vitro decapping rates were assessed in HeLa extractsusing the decapping assay outlined above. RNAs that contained thealpha-globin element showed at least a 2-fold and up to a 10 folddecrease in decapping efficiency compared to control RNAs (FIG. 9).Furthermore, the alpha-globin element also repressed decapping of RNAsubstrates that contained an AU-rich instability element, showing thatthe alpha-globin element exerted a dominant influence over the AU-richinstability element. In order to evaluate whether proteins play a rolein the repression of decapping by the alpha-globin element, competitionassays were performed. The addition of unlabeled competitor RNAs thatcontained the alpha-globin element specifically restored decappingefficiency to wild-type rates. In summary, these data demonstrate thatproteins that bind to the alpha-globin element can stabilize mRNAsthrough a novel mechanism, either by regulating accessibility of the capor interacting directly with factors involved in decapping.

Isolation of protein factors that repress decapping using the standardapproaches to identify RNA binding proteins (i.e. UV crosslinking,mobility shifts in combination with conventional affinitychromatography, northwestern screening, three hybrid assays, etc., iscontemplated, as well as isolation of the polynucleotides encoding thisprotein, and antibodies to this protein, also as described above.

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1-13. (canceled)
 14. A polypeptide which has a molecular weight of about50 to about 100 kilodaltons (kD) in molecular exclusion chromatography,precipitates with 20% ammonium sulfate, elutes at between about 440 to500 mM NaCl from a heparin-Sepharose column, and decaps mammalian RNA.15. A polynucleotide which encodes a polypeptide which has a molecularweight of about 50 to about 100 kilodaltons (kD) in molecular exclusionchromatography, precipitates with 20% ammonium sulfate, elutes atbetween about 440 to 500 mM NaCl from a heparin-Sepharose column, anddecaps mammalian RNA.
 16. An antibody which binds specifically and withhigh affinity to a polypeptide which has a molecular weight of about 50to about 100 kilodaltons (kD) in molecular exclusion chromatography,precipitates with 20% ammonium sulfate, elutes at between about 440 to500 mM NaCl from a heparin-Sepharose column, and decaps mammalian RNA.17-21. (canceled)
 22. A method for carrying out in vitro mammalian mRNAdecapping comprising the steps of: a) providing the composition of claim1, b) incubating said composition at about 30° C. for about 30 min. andmonitoring decapping by detection of release of label from saidcap-labeled RNA.
 23. A method for identifying a compound as a modulatorof mammalian mRNA decapping comprising carrying out the method of claim22 in the presence and absence of said compound, and correlating anychange in decapping by the presence of said compound with modulatoractivity of said compound.
 24. The method of claim 23 wherein saidcap-labeled mRNA substrate comprises poly(A) or at least one RNAelement.
 25. The composition of claim 23 wherein said RNA element is anAU-rich element.
 26. The composition of claim 23 wherein said RNAelement is a pyrimidine-rich element.